Currently there are no successful vaccines for the prevention of feline immunodeficiency disease (FIV) and feline infectious peritonitis (FIP) disease in cats. Current feline leukemia virus (FeLV) vaccines are available, but their level of efficacy remains questionable and, in some cases, may cause the disease. Therefore, there is a need in the art for agents and compositions that provide protection from these and other diseases where there is not yet an existing vaccine or that improve the efficacy of existing and commonly used vaccines. In addition, vaccination of kittens is difficult due to the inability to overcome maternal antibodies in the kittens. Safe and effective agents to help overcome these barriers are also needed. Protection against FIV, FeLV and FIP infection has been correlated with the induction of cytotoxic T cell responses. Anti-FIV and FeLV antibodies have also been shown to be important for successful clearance of these viruses. However, in some cases, anti-FIV envelope antibodies have been implicated in causing enhancement of disease. Anti-FIP spike (Env) antibodies have also been correlated with the induction of enhanced disease and early death after challenge of kittens immunized with vaccines containing FIP spike proteins. A dominant Th2 T cell response has also been observed in the progression of HIV disease. Therefore, vaccines or gene therapies, which induce a strong CTL response are needed.
Nearly one-half of the households in the U.S. have at least one companion animal. In the U.S. alone, there are 55 million dogs and 60 million cats. Cancer is the number one cause of death in dogs and number two cause of death in cats. In one large necropsy study, 23% of dogs died of cancer regardless of age and 43% died of cancer after 10 years of age. 26% of cats died of cancer. Dog and cat cancers are currently treated by resection/amputation, radiation and chemotherapy. Human chemotherapy drugs are used off-label. Combination therapy is common. However, in many cases, treatment regimes are highly toxic, often ineffective and palliative, at best. (Vail D M, MacEwen E G., Cancer Investigation, 18[8]: 781 (2000). MacEwen E G. Presentation given at Biologics for Cancer Diagnosis, Prevention and Immunotherapy Meeting, Ames, Iowa, Apr. 12-13, 2001.) Novel approaches involving the use of biological molecules, such as Eta-1, presented here, have potential for being non-toxic, effective alternatives to standard tumor therapies. There are no cancer treatments currently available, registered specifically for animal use.
Common cat cancers are lymphoma/leukemia, fibrosarcomas, mammary carcinoma, soft tissue sarcoma, and oral squamous cell cancers. Common dog cancers are melanoma, mast cell tumors, soft tissue sarcoma, oral cancers, squamous cell carcinoma, osteosarcoma, and non-hodgkins lymphomas.
Cytotoxic T lymphocytes are the primary effectors of an anti-cancer immune response, mediated by direct and indirect tumor cell killing, or through the release of cytokines capable of interfering with tumor cell growth. Antibodies have a minor role in the rejection of cancer cells. Tumor cells are classified as foreign antigens because they express tumor associated antigens not present in other adult tissues. Still, malignant cells are not recognized by the immune system. Biological agents that increase cytotoxic T cell responses and the production of Th1 cytokines are predicted to have significant potential for cancer treatment as well as for vaccination against cancer.
The Th1/Th2 paradigm predicts that early expression of Th1 cytokines (IFNγ, IL-12, IL-2, TNF) is critical in the generation of a protective immune response against intracellular pathogens, such as viruses and induction of cytotoxic T killer effector cells. Factors augmenting Th1 and inhibiting Th2 cytokine expression (IL-10, IL-4, IL-5) might function as powerful modulators of cell-mediated immunity. The development of cell-mediated (type-1) immune responses is necessary for protection against the growth of many infectious pathogens and can mediate autoimmune host tissue destruction. An essential early step in macrophage activation by microbial pathogens and foreign body reactions is macrophage production of IL-12 at sites of infection, whereas early IL-10 production inhibits this response. Although IL-12 responses can be triggered by an interaction between the CD40 ligand on activated T cells and CD40 on macrophages, this interaction also induces the inhibitory IL-10 cytokine, and its transient nature may not suffice for sustained IL-12 induction in vitro or in vivo.
A gene product that may play an important role in the development of type-1 immunity is the T cell cytokine Eta-1 (for early T lymphocyte activation-1), also known as osteopontin. Eta-1 mRNA is the most abundant early RNA transcript induced in Con-A activated murine T cells (Senger et al., 1979, Cell, 16: 885). Eta-1 is a 60 kDa secreted phosphoprotein containing an RGD domain. The RGD sequence contained in Eta-1 is an integrin-binding motif common to many extracellular matrix (ECM) proteins. The Eta-1 gene is expressed in T cells early in the course of bacterial infections (within 48 hours), and interaction of its protein product with macrophages can induce inflammatory responses. Genetic resistance to infection by certain strains of Rickettsia may depend on Eta-1-dependent attraction of monocytes into infectious sites and acquisition of bacteriocidal activity. Furthermore, the granulomatous responses characteristic of sarcoidosis and tuberculosis are associated with high levels of Eta-1 expression.
Eta-1 enhances Th1 and inhibits Th2 cytokine expression. It directly induces macrophages to induce the production of IL-12, and inhibits IL-10 production. Eta-1 also costimulates T cell proliferation. Eta-1 increases CD3-mediated T-cell production of interferon gamma and CD40 ligand, which augments T-cell dependent IL-12 production by human monocytes. Eta-1 can also induce proliferation of B-cells and antibody production through the involvement of CD40L of B cells. Eta-1 is chemoattractant and supports adhesion of human and murine T cells and macrophages in vitro. In vivo, macrophages accumulate at sites of subcutaneous injection of Eta-1. In addition, Eta-1 may directly induce chemotaxis and indirectly facilitate macrophage migration to other chemoattractants. In vitro migratory and adhesive effects of Eta-1 are mediated by RGD-dependent (integrin) and RGD-independent (CD44) receptors. Eta-1 deficient mice exhibit a five-fold reduction in macrophage infiltration compared with wild-type controls following renal injury. Further, subcutaneous injection of polyvinylpyrrolidone (PVP) induces macrophage-rich granulomas and cellular accumulation after PVP injection is markedly reduced in Eta-1 deficient mice.
Ashkar et al., “Eta-1 (Osteopontin): An Early Component of Type-1 (Cell-Mediated) Immunity”, Science, 287: 860-64 (2000), found that mice deficient in Eta-1 gene expression have severely impaired type-1 immunity to viral infection and bacterial infection and do not develop sarcoid-type granulomas. Eta-1 deficient mice also exhibited diminished IL-12 and interferon-γ production and increased IL-10 production. Ashkar et al. also observed a phosphorylation dependent interaction between the amino-terminal portion of Eta-1 and its integrin receptor mediated stimulation of IL-12 expression and a phosphorylation independent interaction with CD44 mediated inhibition of IL-10 expression. Based on these observations, Ashkar et al. concluded that Eta-1 is a key cytokine that sets the stage for efficient type-1 immune responses through differential regulation of macrophage IL-12 and IL-10 expression.
Eta-1 has also been implicated in various other events that are important to cell-mediated immunity. For example, Eta-1 is associated with monocyte-macrophage differentiation, giant cell formation and the inhibition of apoptosis in various cell types, including pro-B cells. Eta-1 also inhibits nitric oxide production by macrophages and has been associated with tissue repair, fibrosis and dystrophic calcification after immunological injury.
These observations support the idea that Eta-1 polypeptides may be used to initiate or enhance an immune response to an immunogen (viral, bacterial or tumor) in an animal, e.g., by supporting adhesion of T cells and macrophages. Such molecules have use as adjuvants in immunogenic compositions, as well as, e.g., in treating animals suffering from a condition caused by inappropriate altered T cell or macrophage response.
While Eta-1 has been shown to have a recently identified pro-inflammatory role in Th1-mediated immunity, it also appears to have anti-inflammatory effects in different pathological responses. Eta-1 appears to have a role in aberrant tissue repair, has been associated with ras oncogenic transformation of cells (Wu et al. 2000, Brit. J. Cancer, 83: 156-163) and is involved in interactions with CD44 and the formation of metastasis (Weber et al., 1997. Proc. Assoc. Amer. Phys. 109: 1-9). Therefore, Eta appears to have heterogeneic function, and its precise roles in vivo are not completely understood. The multifunctional nature of Eta-1 may reflect the expression of specific isoforms, its secretion as a soluble protein, the location and timing of secretion, and the co-expression of other regulatory factors. Therefore, Eta-1, if appropriately administered, may have multiple applications for use in the treatment of disease (viral, bacterial or tumorigenic). In addition for use as a biological vaccine adjuvant to enhance cell mediated immunity and treatment of cancer, Eta-1 may have use in the treatment of autoimmune diseases, such as osteoarthritis, rheumatoid arthritis, diabetes, and skin diseases.